Binding of integrin aIIbB3 to the RGD ligands, fibrinogen or von Willebrand factor, is necessary for platelet aggregation in hemostasis and arterial thrombosis. The affinity of aIIbB3 for these ligands is low in resting platelets and high in activated ones, indicating intracellular regulation of affinity state. Furthermore, ligand binding to aIIbB3 in platelets and to avB3 in endothelial cells triggers inward signaling, which influences cytoskeletal organization and other cellular responses. The goal of this proposal is to further define the mechanisms of this bidirectional, adhesive signaling by the B3 integrins. Toward this end, two novel and specific interactions between B3 integrins and other proteins will be examined. The first involves the B3 cytoplasmic tail and B3-endonexin (B3-EN), a 12.6 Kda polypeptide that is expressed in several cell types, including platelets and endothelial cells. In order to define the role of B3-EN in integrin function, a mutational analysis of this polypeptide and the B3 tail will be carried out in yeast and mammalian expression systems to map sites of interaction. Also, B3-EN and aIIbB3 will be expressed in Chinese hamster ovary cells to study effects of B3-EN on adhesive signaling. The phosphorylation state of B3-EN and its sub-cellular distribution relative to that of aIIbB and avB3 will be determined in resting and activated platelets and endothelial cells. The second protein interaction to be studied involves aIibB3 or avB3 and derivatives of the fibrinogen-mimetic antibody, PAC1. Native PAC1 contains an RGD integrin recognition motif that is specific for the high affinity form of aIIbB3. In order to develop the tools to directly compare how cells regulate the two B3 integrins, the recognition specificity of PAC1 Fab will be switched from aIIbB3 to avB3 using phage display techniques. The new Fabs will be expressed in insect cells and used to assess how endothelial cells and other cells regulate the affinity state of avB3. Then, aIIbB3 and avB3 will be expressed in a melanoma cell line that contains no endogenous B3 integrins, thus permitting a direct comparison of their affinity states and capacities for inward signaling. Taken together, these studies will provide new information about mechanisms of adhesive signaling by B3 integrins. Furthermore, they should clarify the basis of platelet dysfunction in several hemostatic disorders and suggest improved ways to specifically block formation of platelet thrombi in arterial diseases.